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Fluorescence imaging of mitochondria in cultured skin fibroblasts: a useful method for the detection of oxidative phosphorylation defects.
Tijdschriftbijdrage - Tijdschriftartikel
BACKGROUND:
Protons are pumped from the mitochondrial matrix via oxidative phosphorylation (OXPHOS) into the intermembrane space, creating an electric membrane potential (??) that is used for adenosine triphosphate (ATP) production. Defects in one or more of the OXPHOS complexes are associated with a variety of clinical symptoms, often making it difficult to pinpoint the causal mutation.
METHODS:
In this article, a microscopic method for the quantitative evaluation of ?? in cultured skin fibroblasts is described. The method using 5,5',6,6'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescence staining was tested in a selection of OXPHOS-deficient cell lines.
RESULTS:
A significant reduction of ?? was found in the cell lines of patients with either an isolated defect in complex I, II, or IV or a combined defect (complex I + complex IV). ?? was not reduced in the fibroblasts of two patients with severe complex V deficiency. Addition of the complex I inhibitor rotenone induced a significant reduction of ?? and perinuclear relocalization of the mitochondria. In cells with a heteroplasmic mitochondrial DNA (mtDNA) defect, a more heterogeneous reduction of ?? was detected.
CONCLUSION:
Our data show that imaging of ?? in cultured skin fibroblasts
Protons are pumped from the mitochondrial matrix via oxidative phosphorylation (OXPHOS) into the intermembrane space, creating an electric membrane potential (??) that is used for adenosine triphosphate (ATP) production. Defects in one or more of the OXPHOS complexes are associated with a variety of clinical symptoms, often making it difficult to pinpoint the causal mutation.
METHODS:
In this article, a microscopic method for the quantitative evaluation of ?? in cultured skin fibroblasts is described. The method using 5,5',6,6'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescence staining was tested in a selection of OXPHOS-deficient cell lines.
RESULTS:
A significant reduction of ?? was found in the cell lines of patients with either an isolated defect in complex I, II, or IV or a combined defect (complex I + complex IV). ?? was not reduced in the fibroblasts of two patients with severe complex V deficiency. Addition of the complex I inhibitor rotenone induced a significant reduction of ?? and perinuclear relocalization of the mitochondria. In cells with a heteroplasmic mitochondrial DNA (mtDNA) defect, a more heterogeneous reduction of ?? was detected.
CONCLUSION:
Our data show that imaging of ?? in cultured skin fibroblasts
Tijdschrift: Pediatr Res
ISSN: 0031-3998
Issue: sep
Volume: 72
Pagina's: 232-240
Jaar van publicatie:2012
Trefwoorden:OXPHOS, oxidative phosphorylation, mitochondrial DNA